Mycobacterium tuberculosis is an opportunistic pathogen that causes TB, which is responsible for significant morbidity and mortality worldwide. The emergence of multi-drug resistance (MDR) strains and the AIDS-associated TB infection has led to a critical need for the development of new and effective therapeutic agents for the TB treatment. Adenosine kinase (AK) is a key enzyme in the purine salvage pathway of M. tuberculosis and has recently been identified to be responsible for the bioactivation of a group of nucleoside analogs that are active against M. tuberculosis. Our long term goal is to develop new antimycobacterial agents by exploiting specific inhibition of the purine salvage pathway in M. tuberculosis. We hypothesize that selective inhibition of M. tuberculosis AK or specific activation of nucleoside analogs by the enzyme can be exploited for therapeutic intervention of MDR M. tuberculosis infection. This hypothesis is based on the following observations. 1) Purified M. tuberculosis AK phosphorylated a group of adenosine analogs, including 2-methyladenosine, and the phosphorylation is required for their activation to become selectively active against M. tuberculosis. 2) M. tuberculosis AK share less than 20% sequence identity with the human homolog;therefore, its structure is likely to be significantly different from that of human AK. In addition, the M. tuberculosis purine salvage pathway is not the target of any current TB therapy;therefore, inhibition of this pathway may prove to be a novel therapeutic intervention to MDR-TB. Based on these observations, the experimental focus of this proposal is to unravel the three-dimensional structure of M. tuberculosis AK, explore the structure function relationship of the enzyme, and exploit this relationship for the development of new antimycobacterial agents. We have recently determined the crystal structure of M. tuberculosis AK in complex with adenosine. The structure of this unique bacterial AK revealed a functional dimer structure, the active site conformation and significant structural differences in the active sites between the pathogen and human AK. Our specific aims of this proposed research are: 1). to determine the crystal structure of M. tuberculosis AK in different forms for understanding the enzyme reaction mechanism involved in adenosine phosphorylation and bioactivation of adenosine analogs in M. tuberculosis. 2) To identify M. tuberculosis -specific substrates and inhibitors of AK using a combination of structure- and activity-based approaches. 3) To evaluate the biological activities of the lead compounds and determine the complex structure of M. tuberculosis AK with a lead adenosine analog. These studies will provide molecular basis for development of anti-mycobacterial nucleoside analogs.